Understanding the role of conformational dynamics in protein function has become an increasingly central goal of structural biology. While 53,000 entries in the Protein Data Bank demonstrate the successes in determining the conformational ground state of proteins, the challenges in experimentally characterizing the structure and population of transient conformations form a major impediment to advancement in this field. Hydrogen exchange offers a sensitive monitor of such transient conformations. However, such data is commonly analyzed assuming that solvent-exposed amide hydrogens exchange with the bulk water phase at model peptide rates and are thus insensitive to the residual tertiary structure in the exchange-competent conformation. We have recently reported that protein amide hydrogens which are solvent-exposed in their high resolution X-ray structures exhibit hydroxide-catalyzed exchange rates that deviate from model peptide values by a range of at least three billion-fold. Furthermore, these exchange rates are predictable by standard continuum dielectric Poisson-Boltzmann methods. Applying a chemical interpretation to the hydrogen exchange reaction can yield not only more accurate estimates for the free energy of forming the exchange-competent conformations, it can offer insight into the 3D structure of these transient states. The first two specific aims of this proposal develop two parallel approaches to hydrogen exchange analysis depending upon whether or not an independent prediction of the Boltzmann distribution of protein conformers can be obtained. When such a prediction is impractical for the more structurally protected amides, the exchange-competent conformations will be modeled as constrained by reaction chemistry requirements and, as applicable, by experimental constraints obtained from modulating the electrostatic potential across the protein interior by varying the metal charge of the active site metal. The third specific aim will analyze the intrinsic flexibility and the spatial propagation of ligand-induced changes in flexibility for the immunophilin FK506 binding protein FKBP-12 and the PDZ domains of syntenin which directly participate in melanoma metastasis. These studies will combine the insights gained from the first two aims with the systematic design of hybrid protein structures that exhibit differences in flexibility while preserving parental-like ground state conformational interactions. The increased understanding of the dynamical properties of these two protein systems can assist in the ongoing development of pharmaceuticals directed toward these targets for the treatment of heart failure and cancer metastasis. PUBLIC HEALTH RELEVANCE: Recent scientific advances demonstrate the importance of the intrinsic motions that occur within proteins that shape their biological functions. Our studies develop a novel approach to characterizing these motions within the pharmaceutical targets FKBP12 and the PDZ domains of syntenin which play central roles in acute heart failure and melanoma metastasis, respectively. The increased understanding of these proteins that will arise from this research will provide a broader foundation for the development of clinical therapies for these pathologies.